Flow Cytometry Basic Training

A Look Inside the Box

Himani Anand
Molecular & Cellular
Endocrinology Lab
NIHFW, New Delhi
October 2013

Section I

Background Information on Flow Cytometry

What Is Flow Cytometry?

Flow

~ cells in motion
Cyto ~ cell
Metry ~ measure
Measuring properties of cells while in a
fluid stream

Cytometry vs. Flow Cytometry

Cytometry
Localization of antigen
is possible
Poor enumeration of
cell subtypes
Limiting number of
simultaneous
measurements

Flow Cytometry.
Cannot tell you where
antigen is.
Can analyze many
cells in a short time
frame.
Can look at numerous
parameters at once.

Uses of Flow Cytometry

It can be used for

Immunophenotyping
DNA cell cycle/tumor ploidy
Membrane potential
Ion flux
Cell viability
Intracellular protein staining
pH changes
Cell tracking and proliferation
Sorting
Redox state
Chromatin structure
Total protein
Lipids
Surface charge
Membrane fusion/runover
Enzyme activity
Oxidative metabolism
Sulfhydryl groups/glutathione
DNA synthesis
DNA degradation
Gene expression

The use of flow in research has boomed since the

mid-1980s

Background Info Summary

Flow

Cytometry is a quickly expanding

technology
Has continually increased in popularity
since the mid 1980s.
Gives us the ability to analyze many
properties of many cells in very little time

Section II
The 4 Main Components of a Flow Cytometer

What Happens in a Flow

Cytometer?
Cells in suspension flow single file past
a focused laser where they scatter light and

emit fluorescence that is filtered and

collected
then converted to digitized values that are
stored in a file
Which can then be read by specialized
software.

Fluidics

Interrogation

Electronics

Interpretation

What Happens in a Flow

Cytometer (Simplified)

cellflas h.s wf

The Fluidics System

Cells in suspension flow single file
You

need to have the cells flow one-by-one

into the cytometer to do single cell analysis
Accomplished through a pressurized
laminar flow system.
The sample is injected into a sheath fluid as
it passes through a small orifice (50um300um)

Fluidics Schematic
Sample
Tube

Waste
Tank

Sheath
Tank
Vacuum

Sheath
Pressure

Sample
Pressure
(Variable)

(Constant)

Line Pressure

How The Flow Cell Works

The

cells from the sample tube are injected

into the sheath stream
Flow in a flow cell is laminar.
Hydrodynamic focusing pushes the cells to
line up single file along their long axis.
The shape of the flow cell provides the
means for hydrodynamic focusing.

The Flow Cell

Sheath
Cell

Sample Stream

The introduction of a
large volume into a
small volume in such a
way that it becomes
focused along an axis
is called Hydrodynamic
Focusing.

Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Sample

Sample

Sheath
Sheath

Laser Focal Point

Sheath

Sample
Core
Stream

Incoming Laser

Low Differential

High Differential

Sample Differential
10 psi
10.2 psi

10 psi
10.4 psi

10 psi

10.8 psi

in pressure between sample and sheath

Difference
will control sample volume flow rate
This
greater the differential, the wider the sample core.
The
differential is too large, cells will no longer line up single file
IfResults
wider CVs and increase in multiple cells passing
through thein laser
at once. No more single cell analysis!

Fluidics Recap
Purpose

is to have cells flow one-by-one

past a light source.
Cells move out of tube because there is
slightly greater pressure on the sample than
on the sheath
Cells are focused due to hydrodynamic
focusing and laminar flow.

What Happens in a Flow

Cytometer?
Cells in suspension flow single file past
a focused laser where they scatter light and

emit fluorescence that is filtered, collected

and converted to digitized values that are
stored in a file
Which can then be read by specialized
software.

Fluidics

Interrogation

Electronics

Interpretation

Interrogation
Light

source needs to be focused on the

same point where cells are focused.

Light
On

source
all flow lab instruments-Lasers

Lasers

Light amplification by stimulated emission of radiation

Lasers can provide a single wavelength of light

(monochromatic)
They can provide milliwatts to watts of power
Also provide coherent light
All help to create a stable and reliable signal

Coherent: all emmiting photons have same

wavelength, phase and direction as stimulation
photons

Light Scatter
When

light from a
laser interrogates a
cell, that cell scatters
light in all directions.
The scattered light can
travel from the
interrogation point
down a path to a
detector.

cellflas h.s wf

Forward Scatter
Light

that is scattered in the forward

direction (along the same axis the
laser is traveling) is detected in the
Forward Scatter Channel.
The intensity of this signal has been
attributed to cell size, refractive
index (membrane permeability)
Forward Scatter=
Scatter FSC=FALS=LALS
FSC

Forward Scatter

Laser Beam

Original from Purdue University Cytometry Laboratories

FSC
Detector

Side Scatter
Laser light that

is scattered at 90 degrees to
the axis of the laser path is detected in the
Side Scatter Channel
The intensity of this signal is proportional to
the amount of cytosolic structure in the cell
(eg. granules, cell inclusions, etc.)
Side Scatter=
degree Scatter
Scatter SSC=RALS=90
SSC

Side Scatter
Laser Beam
FSC
Detector

Collection
Lens
SSC
Detector
Original from Purdue University Cytometry Laboratories

Why Look at FSC v. SSC

Since

FSC ~ size and SSC ~ internal structure, a

correlated measurement between them can allow for
differentiation of cell types in a heterogenous cell
population

Granulocytes
SSC

Lymphocytes

Monocytes

RBCs, Debris,
Dead Cells
FSC

Fluorescence Channels
As

the laser interrogates the cell, fluorochromes

on/in the cell (intrinsic or extrinsic) may absorb
some of the light and become excited
As those fluorochromes leave their excited state,
they release energy in the form of a photon with a
specific wavelength, longer than the excitation
wavelength
Those photons pass through the collection lens and
are split and steered down specific channels with
the use of filters.

Fluorescence Detectors
Laser Beam
FSC
Detector

Collection
Lens
Fluorescence
Detector A, B, C, etc
Original from Purdue University Cytometry Laboratories, Modified by James Marvin

Filters
Many

wavelengths of light will be scattered from a cell,

we need a way to split the light into its specific
wavelengths in order to detect them independently. This
is done with filters
Optical filters are designed such that they absorb or
reflect some wavelengths of light, while transmitting
other.
3 types of filters

Long Pass filter

Short Pass filter
Band Pass filter

Long Pass Filters

Transmit

all wavelengths greater than

specified wavelength
500LP will transmit all wavelengths
greater than 500nm
Transmittance

Example:

400nm

500nm

600nm

700nm

Original from Cytomation Training Manual, Modified by James Marvin

Short Pass Filter

Transmits

all wavelengths less than

specified wavelength
600SP will transmit all wavelengths
less than 600nm.
Transmittance

Example:

400nm

500nm

600nm

700nm

Original from Cytomation Training Manual, Modified by James Marvin

Band Pass Filter

Transmits

a specific band of wavelengths

550/20BP Filter will transmit

wavelengths of light between 540nm and
560nm (550/20 = 550+/-10, not 550+/-20)
Transmittance

Example:

400nm

500nm

600nm

700nm

Original from Cytomation Training Manual, Modified by James Marvin

Dichroic Filters
Can

be a long pass or short pass filter

Filter is placed at a 45 angle to the incident light
Part of the light is reflected at 90 to the incident
light, and part of the light is transmitted and
continues on.
Detector 1

Detector 2
Dichroic
Filter

Detectors
There

are two main types of photo detectors

used in flow cytometry
Photodiodes
o

Used for strong signals, when saturation is a

potential problem (eg. FSC detector)

Photomultiplier tubes (PMT)

o

More sensitive than a Photodiode, a PMT is used for

detecting small amounts of fluorescence emitted
from fluorochromes.

Photodiodes and PMTs

Photo

Detectors usually have a band pass

filter in front of them to only allow a
specific band width of light to reach it
Therefore, each detector has a range of light
it can detect, once a filter has been placed in
front of it.

Interrogation Recap

A focused light source (laser) interrogates a cell and scatters

light
That scattered light travels down a channel to a detector
FSC ~ size and cell membrane shape
SSC ~ internal cytosolic structure
Fluorochromes on/in the cell will become excited by the
laser and emit photons
These photons travel down channels and are steered and
split by dichroic (LP/SP) filters
Specific wavelengths are then detected by PMTs that have a
filter in front of them

What Happens in a Flow

Cytometer?
Cells in suspension flow single file past
a focused laser where they scatter light and

emit fluorescence that is collected, filtered

and converted to digitized values that are
stored in a file
Which can then be read by specialized
software.

Fluidics

Interrogation

Electronics

Interpretation

Electronics
Detectors

basically collect photons of light

and convert them to current
The electronics must process that light
signal and convert the current to a digitized
value/# that the computer can graph

What Happens in the PMT

A voltage

is applied to the detector which makes

electrons available for the photons to pick up
As the number of photons increase, more and
more electrons are picked up yielding a greater
current output from the detector
Also, as the voltage applied to the detector
increases the same amount of photons will have a
greater current output

Detector
Current out Photons in

Linear
Amplification

or

Voltage

Voltage

Electronics Schematic

Log
Amplification

Photons

PMT Voltage Input

150V-999V

Original from Becton Dickinson Training manual, Modified by James Marvin

Time

Threshold
When

the laser interrogates an object, light

is scattered.
If the amount of light scattered surpasses a
threshold, then the electronics opens a set
window of time for signal detection
The threshold can be set on any parameter,
but is usually set on FSC

Threshold
FSC
Detector

Threshold
(eg. 52)
Time
FSC
Detector

Threshold
(eg. 52)
Time

Photons In ~ Voltage Out

Photons converted
to no. of electrons

Current out Photons in

Linear
Amplification

or

Voltage

Voltage

Detector

Log
Amplification

PMT Voltage Input

150V-999V

Original from Becton Dickinson Training manual, Modified by James Marvin

Time

The Voltage Pulse

As

the cell passes through the laser, more

and more light is scattered until the cell is in
the center of the laser (maxima)
As the cell leaves the laser, less and less
light is scattered
After a set amount of time, the window
closes until another object scatters enough
light to be triggered.

 Photons/Detector
(V)

The Pulse

Time

Electronics Recap
The

varying number of photons reaching the detector

are converted to a proportional number of electrons
The number of electrons exiting a PMT can be
multiplied by making more electrons available to the
detector (increase Voltage input)
The current generated goes to a log or linear amplifier
where it is amplified (if desired) and is converted to a
voltage pulse
The voltage pulse goes to the ADC to be digitized
The values are placed into a List Mode File

What Happens in a Flow

Cytometer?
Cells in suspension flow single file past
a focused laser where they scatter light and

emit fluorescence that is collected, filtered

and converted to digitized values that are
stored in a file
Which can then be read by specialized
software.

Fluidics

Interrogation

Electronics

Interpretation

Interpretation
Once

the values for each parameter are in a list

mode file, specialized software can graphically
represent it.
The data can be displayed in 1, 2, or 3 dimensional
format
Common programs include

CellQuest
Flowjo
WinMDI
FCS Express

Types of Plots
Single

Color Histogram

Fluorescence intensity (FI) versus count

Two

Color Dot Plot

FI of parameter 1 versus FI of Parameter 2

Two

Color Contour Plot

FI of P1 versus FI of P2. Concentric rings form around

populations. The more dense the population, the closer the

rings are to each other

Two

Color Density Plot

FI of P1 versus FI of P2. Areas of higher density will have a

different color than other areas

Plots
Contour Plot

Density Plot

Greyscale Density

Dot Plot

www.treestar.com

Gating
Is

used to isolate a subset of cells on a plot

Allows the ability to look at parameters
specific to only that subset
Can use boolean logic to include or exclude
multiple gates

Gating Example

Important Points on Analysis

What

How much fluorescence?

What percent are positive?
How much more positive is x than y?
What is the ratio between param1 and param2

What

kind of data are you looking for?

kind of statistics are available

MFI (geometric or arithmetic)

%-ages
CV
Median
Anything you can do with a list of numbers

Everythings Relative
The

relative bin numbers are just that

relative.
Saying your cells have a mean fluorescence
intensity of 100 means absolutely nothing
until you compare it to a negative.
The fact that everything is relative allows
you to compare 2, 3, or 20 samples using
the same instrument settings.

What Happens in a Flow

Cytometer?
Cells in suspension flow single file past
a focused laser where they scatter light and

emit fluorescence that is collected, filtered

and converted to digitized values that are
stored in a file
Which can then be read by specialized
software.

Fluidics

Interrogation

Electronics

Interpretation

References
Numerous

References available in the Flow Lab

Cytometry
Current Protocols in Flow Cytometry
Many more reference books available

Purdue

University Cytometry Laboratories website:

http://www.cyto.purdue.edu/

Dr. Robert Murphy, Carnegie Mellon University- Basic

Theory 1 and 2 powerpoint slides

The

Scripps Research Institute Flow Cytometry

Core Facility: http://facs.scripps.edu/